EP1854874A1 - Method and device for injecting a solution into a cell - Google Patents
Method and device for injecting a solution into a cell Download PDFInfo
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- EP1854874A1 EP1854874A1 EP20070105503 EP07105503A EP1854874A1 EP 1854874 A1 EP1854874 A1 EP 1854874A1 EP 20070105503 EP20070105503 EP 20070105503 EP 07105503 A EP07105503 A EP 07105503A EP 1854874 A1 EP1854874 A1 EP 1854874A1
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- Prior art keywords
- pressure
- valve
- solution
- capillary
- regulator
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
- G01N35/1016—Control of the volume dispensed or introduced
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T137/00—Fluid handling
- Y10T137/206—Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]
- Y10T137/218—Means to regulate or vary operation of device
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T137/00—Fluid handling
- Y10T137/206—Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]
- Y10T137/218—Means to regulate or vary operation of device
- Y10T137/2202—By movable element
- Y10T137/2218—Means [e.g., valve] in control input
Definitions
- the present invention generally relates to injecting material into cells.
- a microinjection device is used for injecting a solution (usually a medicinal solution) into a cell.
- a solution usually a medicinal solution
- Fig. 6 is an example of a conventional device.
- reference numeral 1 denotes a positive pressure pump
- reference numeral 2 denotes a negative pressure pump
- Reference numeral 3 denotes a regulator that is connected to the positive pressure pump 1 and the negative pressure pump 2 for keeping constant the internal pressures thereof.
- Reference numeral 4 denotes a capillary that discharges a solution into a cell due to the pressure from the regulator 3.
- the capillary 4 is similar to an injection syringe, and has a fine needle at its tip.
- the internal diameter of the tip is 0.5 ⁇ m and the external diameter is 1 ⁇ m.
- Reference numeral 6 denotes a tube that transmits the pressure from the regulator 3 to the capillary 4, and reference numeral 5 denotes a pressure sensor which is located in the middle of the tube 6.
- the pressure detected by the pressure sensor 5 is output to the regulator 3, and the regulator 3 adjusts the internal pressure so that the pressure detected by the pressure sensor 5 is maintained constant. Otherwise, it is possible to arrange the pressure sensor inside the regulator 3 and adjust the internal pressure so as to maintain constant output from the pressure sensor. Electric voltage is input to the regulator 3 as a control signal, and the regulator 3 generates a pressure that is proportional to the input electric voltage.
- the needle attached to the tip of the capillary 4 is filled with the solution.
- Pressure applied by the regulator 3 urges a cylinder of the capillary 4 whereby the solution is discharged (injected) into the cell.
- the cell is observed for a change that occurs after injection of the solution.
- Fig. 7 depicts a pressure response curve of the conventional device.
- a horizontal axis indicates time and a vertical axis indicates pressure.
- the pressure is maintained at a reverse flow preventing pressure.
- the pressure reaches an injection pressure, and the capillary 4 injects the solution into the cell.
- the state in which the capillary 4 is injected into the cell continues for a predetermined period after which the pressure is lowered gradually.
- the injecting operation stops when the pressure reaches the reverse flow preventing pressure.
- the conventional device can be used in a gene delivery device.
- cells that flow through a micro fluid channel are observed by using a cell observing device, cells are trapped one by one with a cell trapping device, and genetic material and medicinal solutions are discharged into the cells by using a gene delivering micro needle (for example, see Japanese Patent Application Laid-Open No. 2004-166653 ).
- a microinjection apparatus has, at its tip, a micro instrument that is connected to a micro syringe, which is filled with the solution. When a male screw is rotated to move a plunger, into the micro syringe, the solution is discharged from the micro instrument (for example, see Japanese Patent Application Laid-Open No. H03-119989 ).
- a microinjecting method of the microinjection apparatus involves filling a predetermined volume of a solution in an extra fine capillary, with a tip that is of ⁇ m order of size, injecting the solution into the cell by pressurizing the capillary, and observing the response. In this process, it is necessary to control sequentially switching of the pressure between a pressure necessary for discharging the solution in the cell and a pressure necessary for preventing reverse flow of the solution into the capillary.
- the conventional microinjection device which has the structure shown in Fig. 6, does not take into account the pressure transient response. Therefore, when trace quantity of solution of pl (picolitre) order is to be discharged through injections such as injections into animal cells, if the delivery time is lowered to less than 1 second for speeding a discharge cycle, the set pressure and the set time deviate from the actual response, and adjustment of discharging volume of the solution is difficult.
- a solution discharging method for discharging a solution, from a hollow capillary with a narrow tip that is filled with the solution, into a cell due to an action of gas pressure includes connecting a regulating chamber to the capillary by a valve; and regulating gas in the regulating chamber at a predetermined pressure before opening and closing the valve so that regulated pressure has a generally rectangular waveform thereby controlling quantity of the solution that is to be injected from the capillary into the cell to be constant.
- a microinjection device includes a pressure pump; a regulator that is connected to the pressure pump and that maintains constant pressure; a regulating chamber that is connected to the regulator and an internal pressure of which is maintained to a predetermined pressure; a valve that is connected to the regulating chamber; and a capillary that is connected to the valve and that is used to inject solution in a cell.
- Fig. 1 is a schematic of a first embodiment of the present invention. Components that are same as those shown in Fig. 6 are indicated with the same reference numerals.
- reference numeral 1 denotes a positive pressure pump
- reference numeral 2 denotes a negative pressure pump
- Reference numeral 3 denotes a regulator that regulates the pressure
- reference numeral 10 denotes a regulating chamber that contains a gas (typically air) whose pressure is regulated by the regulator 3.
- Reference numeral 5 denotes a second pressure sensor that detects pressure in the regulating chamber 10. The output of the second pressure sensor 5 is input in the regulator 3.
- Reference numeral 4 denotes a capillary that injects solution into an animal cell and the like
- reference numeral 11 denotes a valve arranged in between the regulating chamber 10 and the capillary 4.
- the valve 11, the construction of which may include a solenoid for example, is opened and closed to transmit air from the regulating chamber 10 to the capillary 4.
- Reference numeral 12 denotes a pressure sensor 1 that detects pressure of air in the capillary 4.
- the pressure of air inside the capillary 4 is P 1
- the volume is V 1 . Operation of the apparatus, which has such a configuration, is explained below.
- pressure of the regulating chamber 10 is set by the regulator 3 to a certain degree higher than the injection pressure.
- the operator while watching under a microscope, confirms that a needle of the capillary 4 reaches the cell, the operator opens the valve 11 to bring the pressure to injection pressure level.
- the valve 11 is immediately closed. While the capillary 4 is discharging (injecting) the solution into the cell, the regulator 3 adjusts pressure in the regulating chamber 10 and sets it to a lower level than the reverse flow preventing pressure.
- the pressure P after the opening of the valve is set in advance
- the pressure P 1 in the capillary before the opening of the valve is known in advance from an output from the pressure sensor 12; therefore, it is possible to calculate the pressure P 2 of the regulator before the opening of the valve through Equation (3).
- the pressure P 2 of the regulator before the opening of the valve is set to the value derived through Equation (3)
- the pressure at the time of opening of the valve 11 is regulated to the pressure P. That is, it is possible to maintain the pressure in the regulating chamber and the capillary.
- FIG. 7 Pressure response in an ordinary microinjection device is as shown in Fig. 7.
- Fig. 2 is a graph for explaining the pressure response according to the present invention.
- the horizontal axis is time and the vertical axis is the pressure.
- the time interval required for responding to regulation of the pressure to the injection pressure level is still shorter than the response as shown in Fig. 7.
- Fig. 7 in the characteristic of the conventional device, the time required to attain target value is longer, whereas the transient response of the device in the present invention is only represented by vibrations near the target value. If the integration value of the vibrations is considered zero, it can be thought that the total quantity of injecting solution is proportional to a product of the target pressure and the time required for application of the pressure.
- a valve is arranged in between a regulating chamber and a capillary, and opened and closed to control the discharging quantity of solution from the capillary into a cell. That is, when the solution is injected into a cell, the quantity of solution can be easily controlled, and stable microinjection can be performed at high speed with less effect of transient response.
- speedy rise of pressure in the capillary produces a roughly rectangular waveform that results in a high degree of accuracy in time required for application of the pressure, high speed injection cycle, and improved accuracy of the quantity of the injecting solution.
- the quantity of the injecting solution is proportional to pressure and time for which the pressure is applied; therefore, even if there is transient response, a method of controlling the quantity of the injecting solution according to the pressure time integration also has the same effect on the accuracy of the quantity of the injecting solution. That is, according to the present invention, because the integration value of the pressure applied to the capillary and the time for which the pressure is applied is controlled to a predetermined value, it is possible to always control the quantity of the injecting solution to a constant quantity.
- Fig. 3 is a schematic of a second embodiment according to the present invention. Components that are same as those in Fig. 1 are indicated with the same reference numerals.
- the embodiment includes a second valve 15 in between the regulator 3 and the regulating chamber 10. Other aspects of the structure are same as shown in Fig. 1. In such a structure when the first valve 11 is opened and closed, the second valve 15 is kept closed, which prevents pressure fluctuations from being conveyed to the regulator 3 and prevents occurrence of fluctuation in pressure.
- Fig. 4 is a schematic of a third embodiment according to the present invention. Components that are same as those in Fig. 3 are indicated with the same reference numerals.
- reference numeral 25 denotes a second valve, which is connected to the regulator 3;
- reference numeral 20 denotes a second regulating chamber connected to the second valve;
- reference numeral 21 denotes a fourth valve that is connected to the second regulating chamber.
- Reference numeral 22 denotes a pressure sensor that detects a pressure Pi in the first regulating chamber, and reference numeral 23 denotes a pressure sensor that detects a pressure Pc in the second regulating chamber.
- Reference numeral 12 denotes a pressure sensor that detects the pressure in the capillary.
- Reference numeral 15 denotes the first valve
- reference numeral 10 denotes the first regulating chamber connected to the first valve
- reference numeral 11 denotes a third valve connected to the first regulating chamber.
- the first valve and the second valve are commonly connected to the regulator 3, and the third valve and the fourth valve are commonly connected to the capillary 4.
- a double system of regulator units formed of valves regulators, and valves.
- the third valve is closed, and the capillary 4 is injecting the solution in the cell, there is no need to regulate the regulating chamber 10. That is, when one system is operating the capillary 4, another system regulates the regulating chamber, and when injection operation of the capillary 4 is completed, opening of the fourth valve leads to a faster regulation after the opening of the valve.
- time required to switch valves from one state to another is shortened.
- Fig. 5 is a schematic of a fourth embodiment according to the present invention. Components that are same as those in Fig. 4 are indicated with the same reference numerals.
- the embodiment includes a second regulator 30 corresponding to the second regulation system shown in Fig. 4.
- the second regulator 30 is connected to the positive pump 1 and the negative pump 2, and is independent of a first regulator 3.
- the second regulator 30 is connected to the second valve. Remaining structure is the same as that shown in Fig. 4.
- Such a structure allows the regulator 3 and a regulator 30 to adjust the air pressure in the regulator chamber independently, which allows the two systems to operate independently, and this structure operates faster than the one shown in Fig. 4.
- high accuracy of time for which pressure is applied can be achieved due to fast rise in pressure response that creates a generally rectangular waveform, and it is possible to have faster injection cycle and improve accuracy in quantity of discharging solution.
Abstract
Description
- The present invention generally relates to injecting material into cells.
- In the field of new drug designing, a microinjection device is used for injecting a solution (usually a medicinal solution) into a cell. Fig. 6 is an example of a conventional device. As shown in the diagram,
reference numeral 1 denotes a positive pressure pump, andreference numeral 2 denotes a negative pressure pump.Reference numeral 3 denotes a regulator that is connected to thepositive pressure pump 1 and thenegative pressure pump 2 for keeping constant the internal pressures thereof. -
Reference numeral 4 denotes a capillary that discharges a solution into a cell due to the pressure from theregulator 3. Thecapillary 4 is similar to an injection syringe, and has a fine needle at its tip. The internal diameter of the tip is 0.5 µm and the external diameter is 1 µm.Reference numeral 6 denotes a tube that transmits the pressure from theregulator 3 to thecapillary 4, andreference numeral 5 denotes a pressure sensor which is located in the middle of thetube 6. - The pressure detected by the
pressure sensor 5 is output to theregulator 3, and theregulator 3 adjusts the internal pressure so that the pressure detected by thepressure sensor 5 is maintained constant. Otherwise, it is possible to arrange the pressure sensor inside theregulator 3 and adjust the internal pressure so as to maintain constant output from the pressure sensor. Electric voltage is input to theregulator 3 as a control signal, and theregulator 3 generates a pressure that is proportional to the input electric voltage. - The needle attached to the tip of the
capillary 4 is filled with the solution. Pressure applied by theregulator 3 urges a cylinder of thecapillary 4 whereby the solution is discharged (injected) into the cell. The cell is observed for a change that occurs after injection of the solution. When the pressure inside theregulator 3 is to be brought back to the atmospheric pressure, air is pulled out from theregulator 3 by thenegative pressure pump 2 whereby the pressure inside theregulator 3 is quickly brought back to the atmospheric pressure. - Fig. 7 depicts a pressure response curve of the conventional device. A horizontal axis indicates time and a vertical axis indicates pressure. Initially, the pressure is maintained at a reverse flow preventing pressure. After time T1 is elapsed, the pressure reaches an injection pressure, and the
capillary 4 injects the solution into the cell. The state in which thecapillary 4 is injected into the cell continues for a predetermined period after which the pressure is lowered gradually. The injecting operation stops when the pressure reaches the reverse flow preventing pressure. - The conventional device can be used in a gene delivery device. In the gene delivery device, cells that flow through a micro fluid channel are observed by using a cell observing device, cells are trapped one by one with a cell trapping device, and genetic material and medicinal solutions are discharged into the cells by using a gene delivering micro needle (for example, see
Japanese Patent Application Laid-Open No. 2004-166653 Japanese Patent Application Laid-Open No. H03-119989 - A microinjecting method of the microinjection apparatus involves filling a predetermined volume of a solution in an extra fine capillary, with a tip that is of µm order of size, injecting the solution into the cell by pressurizing the capillary, and observing the response. In this process, it is necessary to control sequentially switching of the pressure between a pressure necessary for discharging the solution in the cell and a pressure necessary for preventing reverse flow of the solution into the capillary.
- However, the conventional microinjection device, which has the structure shown in Fig. 6, does not take into account the pressure transient response. Therefore, when trace quantity of solution of pl (picolitre) order is to be discharged through injections such as injections into animal cells, if the delivery time is lowered to less than 1 second for speeding a discharge cycle, the set pressure and the set time deviate from the actual response, and adjustment of discharging volume of the solution is difficult.
- Therefore, using the conventional microinjection apparatus, an experienced operator would adjust the pressure and the time for applying the pressure while taking into account swelling of the cell when injecting the solution into the cell. However, in this method, it is unclear whether a constant amount of material is discharged into the cell.
- It is desirable to at least partially solve the problems in the conventional technology.
- According to an aspect of the present invention, a solution discharging method for discharging a solution, from a hollow capillary with a narrow tip that is filled with the solution, into a cell due to an action of gas pressure, includes connecting a regulating chamber to the capillary by a valve; and regulating gas in the regulating chamber at a predetermined pressure before opening and closing the valve so that regulated pressure has a generally rectangular waveform thereby controlling quantity of the solution that is to be injected from the capillary into the cell to be constant.
- According to another aspect of the present invention, a microinjection device includes a pressure pump; a regulator that is connected to the pressure pump and that maintains constant pressure; a regulating chamber that is connected to the regulator and an internal pressure of which is maintained to a predetermined pressure; a valve that is connected to the regulating chamber; and a capillary that is connected to the valve and that is used to inject solution in a cell.
- The above and other features, advantages and technical and industrial significance of this invention will be better understood by reading the following detailed description of presently preferred embodiments of the invention, when considered in connection with the accompanying drawings, in which:
- Fig. 1 is a schematic of a first embodiment according to the present invention;
- Fig. 2 is a graph for explaining a pressure response according to the present invention;
- Fig. 3 is a schematic of a second embodiment according to the present invention;
- Fig. 4 is a schematic of a third embodiment according to the present invention;
- Fig. 5 is a schematic of a fourth embodiment according to the present invention;
- Fig. 6 is a schematic of an example of the structure of a conventional device; and
- Fig. 7 is a graph for explaining a pressure response of the conventional device.
- Exemplary embodiments of the present invention are explained below in detail with reference to the accompanying drawings. Fig. 1 is a schematic of a first embodiment of the present invention. Components that are same as those shown in Fig. 6 are indicated with the same reference numerals. As shown in the diagram,
reference numeral 1 denotes a positive pressure pump, andreference numeral 2 denotes a negative pressure pump.Reference numeral 3 denotes a regulator that regulates the pressure,reference numeral 10 denotes a regulating chamber that contains a gas (typically air) whose pressure is regulated by theregulator 3.Reference numeral 5 denotes a second pressure sensor that detects pressure in the regulatingchamber 10. The output of thesecond pressure sensor 5 is input in theregulator 3. - Pressure of air in the regulating
chamber 10 is P2 and volume is V2.Reference numeral 4 denotes a capillary that injects solution into an animal cell and the like,reference numeral 11 denotes a valve arranged in between theregulating chamber 10 and thecapillary 4. Thevalve 11, the construction of which may include a solenoid for example, is opened and closed to transmit air from theregulating chamber 10 to thecapillary 4.Reference numeral 12 denotes apressure sensor 1 that detects pressure of air in thecapillary 4. The pressure of air inside thecapillary 4 is P1, and the volume is V1. Operation of the apparatus, which has such a configuration, is explained below. - First, pressure of the regulating
chamber 10 is set by theregulator 3 to a certain degree higher than the injection pressure. When an operator, while watching under a microscope, confirms that a needle of thecapillary 4 reaches the cell, the operator opens thevalve 11 to bring the pressure to injection pressure level. When the pressure reaches the injection pressure level, thevalve 11 is immediately closed. While thecapillary 4 is discharging (injecting) the solution into the cell, theregulator 3 adjusts pressure in the regulatingchamber 10 and sets it to a lower level than the reverse flow preventing pressure. - Afterwards, when the
valve 11 is opened, pressure in theregulating chamber 10 and thecapillary 4 becomes equal to the reverse flow preventing pressure, which prevents the solution from reverting into thecapillary 4. The regulatingchamber 10 is set to lower than the reverse flow preventing pressure in advance, which makes it possible to bring the level of the pressure entirely to the reverse flow preventing pressure, when thevalve 11 is opened. - The relationship between the pressure before the opening of the valve and the pressure after the opening of the valve can be obtained through an equation of state of air. When P denotes pressure after the opening of the valve, P1 denotes pressure in the capillary before the opening of the valve, P2 denotes pressure in the regulator before the opening of the valve, V1 denotes volume of air in the capillary, and V2 denotes volume of air in the regulator, the pressure P after opening of the valve and volume ratio r\(=V1/V2) are represented with following equations:
Equation 1, the pressure P2 of the regulator before opening of the valve is represented with the following equation:Equation 2. Moreover, the pressure P after the opening of the valve is set in advance, the pressure P1 in the capillary before the opening of the valve is known in advance from an output from thepressure sensor 12; therefore, it is possible to calculate the pressure P2 of the regulator before the opening of the valve through Equation (3). When the pressure P2 of the regulator before the opening of the valve is set to the value derived through Equation (3), the pressure at the time of opening of thevalve 11 is regulated to the pressure P. That is, it is possible to maintain the pressure in the regulating chamber and the capillary. - Pressure response in an ordinary microinjection device is as shown in Fig. 7. On the other hand, when the valve is switched from one state to another state in an instant, the pressure is transmitted at a sonic speed. The pressure response in such a case is as shown in a graph in Fig. 2. Fig. 2 is a graph for explaining the pressure response according to the present invention. The horizontal axis is time and the vertical axis is the pressure.
- When the
valve 11 is opened, the pressure rises from the reverse flow preventing pressure up to the injection pressure. Subsequently, when thevalve 11 is closed, as shown in the diagram, the pressure drops to the reverse flow preventing pressure from the injection pressure in an instant. Rise and fall of the pressure is faster than the characteristic of the conventional device in Fig. 7. - As shown in the diagram, although some degree of transient response occurs due to reflection, the time interval required for responding to regulation of the pressure to the injection pressure level is still shorter than the response as shown in Fig. 7. As shown by Fig. 7 in the characteristic of the conventional device, the time required to attain target value is longer, whereas the transient response of the device in the present invention is only represented by vibrations near the target value. If the integration value of the vibrations is considered zero, it can be thought that the total quantity of injecting solution is proportional to a product of the target pressure and the time required for application of the pressure.
- Thus, according to the first embodiment, a valve is arranged in between a regulating chamber and a capillary, and opened and closed to control the discharging quantity of solution from the capillary into a cell. That is, when the solution is injected into a cell, the quantity of solution can be easily controlled, and stable microinjection can be performed at high speed with less effect of transient response. According to the first embodiment, speedy rise of pressure in the capillary produces a roughly rectangular waveform that results in a high degree of accuracy in time required for application of the pressure, high speed injection cycle, and improved accuracy of the quantity of the injecting solution.
- Generally, the quantity of the injecting solution is proportional to pressure and time for which the pressure is applied; therefore, even if there is transient response, a method of controlling the quantity of the injecting solution according to the pressure time integration also has the same effect on the accuracy of the quantity of the injecting solution. That is, according to the present invention, because the integration value of the pressure applied to the capillary and the time for which the pressure is applied is controlled to a predetermined value, it is possible to always control the quantity of the injecting solution to a constant quantity.
- Fig. 3 is a schematic of a second embodiment according to the present invention. Components that are same as those in Fig. 1 are indicated with the same reference numerals. The embodiment includes a
second valve 15 in between theregulator 3 and the regulatingchamber 10. Other aspects of the structure are same as shown in Fig. 1. In such a structure when thefirst valve 11 is opened and closed, thesecond valve 15 is kept closed, which prevents pressure fluctuations from being conveyed to theregulator 3 and prevents occurrence of fluctuation in pressure. - Fig. 4 is a schematic of a third embodiment according to the present invention. Components that are same as those in Fig. 3 are indicated with the same reference numerals. In the diagram,
reference numeral 25 denotes a second valve, which is connected to theregulator 3;reference numeral 20 denotes a second regulating chamber connected to the second valve; andreference numeral 21 denotes a fourth valve that is connected to the second regulating chamber.Reference numeral 22 denotes a pressure sensor that detects a pressure Pi in the first regulating chamber, andreference numeral 23 denotes a pressure sensor that detects a pressure Pc in the second regulating chamber.Reference numeral 12 denotes a pressure sensor that detects the pressure in the capillary. -
Reference numeral 15 denotes the first valve,reference numeral 10 denotes the first regulating chamber connected to the first valve, andreference numeral 11 denotes a third valve connected to the first regulating chamber. The first valve and the second valve are commonly connected to theregulator 3, and the third valve and the fourth valve are commonly connected to thecapillary 4. - Thus, according to the third embodiment, there is provided a double system of regulator units formed of valves regulators, and valves. In this structure, while the third valve is closed, and the
capillary 4 is injecting the solution in the cell, there is no need to regulate the regulatingchamber 10. That is, when one system is operating thecapillary 4, another system regulates the regulating chamber, and when injection operation of thecapillary 4 is completed, opening of the fourth valve leads to a faster regulation after the opening of the valve. Thus, according to the third embodiment, time required to switch valves from one state to another is shortened. - Fig. 5 is a schematic of a fourth embodiment according to the present invention. Components that are same as those in Fig. 4 are indicated with the same reference numerals. The embodiment includes a
second regulator 30 corresponding to the second regulation system shown in Fig. 4. Thesecond regulator 30 is connected to thepositive pump 1 and thenegative pump 2, and is independent of afirst regulator 3. Thesecond regulator 30 is connected to the second valve. Remaining structure is the same as that shown in Fig. 4. - Such a structure allows the
regulator 3 and aregulator 30 to adjust the air pressure in the regulator chamber independently, which allows the two systems to operate independently, and this structure operates faster than the one shown in Fig. 4. - Thus, according to an aspect of the present invention, high accuracy of time for which pressure is applied can be achieved due to fast rise in pressure response that creates a generally rectangular waveform, and it is possible to have faster injection cycle and improve accuracy in quantity of discharging solution.
- Although the invention has been described with respect to a specific embodiment for a complete and clear disclosure, the appended claims are not to be thus limited but are to be construed as embodying all modifications and alternative constructions that may occur to one skilled in the art that fairly fall within the basic teaching herein set forth.
Claims (7)
- A solution discharging method for discharging a solution, from a hollow capillary with a narrow tip that is filled with the solution, into a cell due to an action of air pressure, the solution discharging method comprising:connecting a regulating chamber to the capillary by a valve; andregulating air in the regulating chamber at a predetermined pressure before opening and closing the valve so that regulated pressure has a rectangular waveform thereby controlling quantity of the solution that is to be injected from the capillary into the cell to be constant.
- The solution discharging method according to claim 1, wherein an integration value of pressure applied to the capillary and time for which the pressure is applied is controlled to a predetermined value thereby controlling the quantity of the solution that is to be injected into the cell.
- A microinjection device comprising:a pressure pump;a regulator that is connected to the pressure pump and that maintains constant pressure;a regulating chamber that is connected to the regulator and an internal pressure of which is maintained to a predetermined pressure;a valve that is connected to the regulating chamber; anda capillary that is connected to the valve and that is used to inject solution in a cell.
- The microinjection device according to claim 3, wherein after the valve is opened, the internal pressure of the regulating chamber is regulated to a lower pressure than a reverse flow preventing pressure by closing the valve while the solution is being injected into the cell.
- The microinjection device according to claim 3, further comprising a second valve that is located between the regulator and the regulating chamber.
- The microinjection device according to claim 3, wherein the regulator includes a double system of regulators to maintain a predetermined pressure that is to be applied to the capillary.
- The microinjection device according to claim 3, wherein the regulator includes a double system of regulators to maintain constant pressure, and the regulating chamber includes a double system of regulating chambers to maintain the internal pressure to a predetermined pressure.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006133512A JP5011812B2 (en) | 2006-05-12 | 2006-05-12 | Method for discharging liquid into cell and microinjection apparatus |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1854874A1 true EP1854874A1 (en) | 2007-11-14 |
EP1854874B1 EP1854874B1 (en) | 2010-06-09 |
Family
ID=38235404
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20070105503 Expired - Fee Related EP1854874B1 (en) | 2006-05-12 | 2007-04-02 | Method and device for injecting a solution into a cell |
Country Status (5)
Country | Link |
---|---|
US (1) | US8012417B2 (en) |
EP (1) | EP1854874B1 (en) |
JP (1) | JP5011812B2 (en) |
KR (1) | KR100870167B1 (en) |
DE (1) | DE602007007011D1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010035457A (en) * | 2008-08-01 | 2010-02-18 | Fujitsu Ltd | Injection device and method of injection by using the same |
JP5272633B2 (en) * | 2008-10-06 | 2013-08-28 | 富士通株式会社 | Injection device |
JP5141543B2 (en) * | 2008-12-25 | 2013-02-13 | 富士通株式会社 | Microinjection apparatus and backflow prevention pressure measuring method |
Citations (5)
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GB2114740A (en) * | 1982-02-05 | 1983-08-24 | Europ Lab Molekularbiolog | Method and apparatus for injecting miniscule quantities of samples into cells |
JPH03119989A (en) | 1989-10-02 | 1991-05-22 | Toshiro Higuchi | Micro-injector and method for controlling injection thereof |
JPH04304881A (en) * | 1991-03-29 | 1992-10-28 | Shimadzu Corp | Equipment for injection |
WO1993007256A1 (en) * | 1991-10-07 | 1993-04-15 | Ciba-Geigy Ag | Particle gun for introducing dna into intact cells |
DE4401076A1 (en) | 1994-01-15 | 1995-07-20 | Eppendorf Geraetebau Netheler | Injection appts. for biological cells |
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JPS62224361A (en) * | 1986-03-26 | 1987-10-02 | アイシン精機株式会社 | Auxiliary circulation machinery driving apparatus |
JP2513359B2 (en) * | 1990-11-30 | 1996-07-03 | 株式会社島津製作所 | Micro pipette device |
US5456880A (en) * | 1992-11-20 | 1995-10-10 | Shimadzu Corporation | Micropipet apparatus and micromanipulator |
JP2003186549A (en) * | 2001-10-12 | 2003-07-04 | Smc Corp | Fluid pressure regulator |
US6779541B2 (en) * | 2001-10-12 | 2004-08-24 | Smc Kabushiki Kaisha | Fluid pressure regulator |
US6958043B2 (en) * | 2002-05-21 | 2005-10-25 | Medtronic Xomed, Inc. | Apparatus and method for displacing the partition between the middle ear and the inner ear using a manually powered device |
JP4278365B2 (en) | 2002-11-22 | 2009-06-10 | 富士通株式会社 | Transduced cell production device |
CN1950124B (en) * | 2004-01-12 | 2010-11-10 | Ⅰ科学外科公司 | Injector for viscous materials |
JP4456429B2 (en) * | 2004-07-27 | 2010-04-28 | 富士通株式会社 | Injection device |
KR100597814B1 (en) * | 2005-10-31 | 2006-07-10 | (주)엠큐어 | Pneumatic injection gun |
-
2006
- 2006-05-12 JP JP2006133512A patent/JP5011812B2/en not_active Expired - Fee Related
-
2007
- 2007-04-02 EP EP20070105503 patent/EP1854874B1/en not_active Expired - Fee Related
- 2007-04-02 DE DE200760007011 patent/DE602007007011D1/en active Active
- 2007-04-25 KR KR1020070040324A patent/KR100870167B1/en active IP Right Grant
- 2007-05-04 US US11/797,630 patent/US8012417B2/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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GB2114740A (en) * | 1982-02-05 | 1983-08-24 | Europ Lab Molekularbiolog | Method and apparatus for injecting miniscule quantities of samples into cells |
JPH03119989A (en) | 1989-10-02 | 1991-05-22 | Toshiro Higuchi | Micro-injector and method for controlling injection thereof |
JPH04304881A (en) * | 1991-03-29 | 1992-10-28 | Shimadzu Corp | Equipment for injection |
WO1993007256A1 (en) * | 1991-10-07 | 1993-04-15 | Ciba-Geigy Ag | Particle gun for introducing dna into intact cells |
DE4401076A1 (en) | 1994-01-15 | 1995-07-20 | Eppendorf Geraetebau Netheler | Injection appts. for biological cells |
Also Published As
Publication number | Publication date |
---|---|
JP2007300868A (en) | 2007-11-22 |
DE602007007011D1 (en) | 2010-07-22 |
KR100870167B1 (en) | 2008-11-25 |
JP5011812B2 (en) | 2012-08-29 |
US8012417B2 (en) | 2011-09-06 |
US20070264162A1 (en) | 2007-11-15 |
EP1854874B1 (en) | 2010-06-09 |
KR20070109832A (en) | 2007-11-15 |
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